Cupredoxin derived transport agents and methods of use thereof

ABSTRACT

The present invention discloses methods and materials for delivering a cargo compound into a cancer cell. Delivery of the cargo compound is accomplished by the use of protein transduction domains derived from cupredoxins. The invention further discloses methods for treating cancer and diagnosing cancer.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §§119 and 120 ofU.S. application Ser. No. 12/754,197, filed Apr. 5, 2010, and issued asU.S. Pat. No. 8,206,685 Jun. 26, 2012, which is a continuation of U.S.application Ser. No. 11/244,105, filed Oct. 6, 2005, and issued as U.S.Pat. No. 7,691,383 Apr. 6, 2010, which claims priority to U.S.Provisional Patent Application No. 60/616,782, filed Oct. 7, 2004, U.S.Provisional Patent Application No. 60/680,500, filed May 13, 2005, andU.S. Provisional Patent Application No. 60/700,297, filed Jul. 19, 2005.The entire content of those applications are fully incorporated hereinby reference.

BACKGROUND

The entry of a protein into a mammalian cell is often dictated by asmall segment of the protein, which is commonly referred to as a“protein transduction domain” or PTD. This segment can be used as asignal attached to a foreign protein to facilitate transport of such aprotein into a mammalian cell. For example, amphipathic peptides areused to facilitate uptake of DNA-cleaving metalloporphyrins as potentialantitumor drugs in human fibroblasts HS68 or murine lymphocytic leukemiaL1210 cells (Chaloin, L. et al. Bioconjugate Chem. 12:691-700, (2001)).Peptides, called cell-penetrating peptides, such as penetratin,transportan, Tat (amino acids 47-57 or 48-60) and the model amphipathicpeptide MAP, have been used as delivery vehicles for transportingpharmacologically important substances, such as antisenseoligonuclotides, proteins and peptides (Hallbrink, M. et al. Biochim.Biophys. Acta 1515:101-109 (2001); Lindgren, M., et al. TrendsPharmacol. Sci. 21:99-103 (2000)).

Such peptides, particularly the DNA-binding homeodomain of Antennapedia,a Drosophila transcription factor, or the 21 residue peptide carrierPep-1, are internalized by many types of cells in culture, such as humanHS68 or murine NIH-3T3 fibroblasts, at either 37° C. or 4° C. The lackof effect of the temperature shift suggests a penetration mechanismdifferent from that of classical endocytosis (Morris, M. C. et al.Nature Biotechnol. 19:1173-1176 (2001)), which requires chiral receptorproteins. One of the most widely used peptides to transportpharmacologically-active compounds in mammalian cells is the elevenamino acid arginine-rich protein transduction domain (PTD) of the humanimmunodeficiency virus type 1 (HIV-1) transactivator protein Tat(Schwarze, S. R. et al. Science 285:1569-1572 (1999), Schwarze, S. R. etal. Trends Cell Biol. 10:290-295 (2000)). Intraperitoneal injection ofthe 120 kDa beta-galactosidase/Tat fusion protein results in thetranscellular transduction of the fusion protein into virtually alltissues in mice, including the passage of the blood-brain barrier. Thisshort peptide domain of HIV-1 Tat has been shown to mediate cellinternalization of large molecules or particles, including magneticnanoparticles, phage vectors, liposomes and plasmid DNA. Unlike theother cell-penetrating peptides discussed above, internalization ofcargo proteins by full length Tat or its 11 amino acid transductiondomain is significantly impaired at 4° C. (Liu, Y. et al. Nat. Med.6:1380-1387 (2000), Suzuki, T. et al. J. Biol. Chem. 277:2437-2443(2002)) and requires interactions with receptors such as the heparansulfate chains of the cell membrane heparan sulfate proteoglycans.

Most of the PTDs identified to date have been derived from viral andmammalian sources. Other sources of PTDs would be desirable for thedesign of various experimental sequences, and for animal and humantherapies and prophylactic procedures. One alternative source of PTDs isbacterial cells. Although bacterial proteins such as cholera toxin areknown to enter mammalian cell cytosol (Sofer, A. and Futerman, A. H. J.Biol. Chem. 270:12117-12122 (1995)), the cytotoxicity of such proteinshas limited the use of bacterial proteins, or PTDs derived from them,for transporting pharmacologically important cargos in mammalian cells.

SUMMARY OF THE INVENTION

One aspect of the present invention is peptide that has at least about90% amino acid sequence identity to less than a full length wild-typecupredoxin or H.8 outer membrane protein, and which facilitates theentry of an linked cargo molecule into a mammalian cancer cell. In someembodiments, the peptide has at least about 90% amino acid sequenceidentity to less than a full length azurin, plastocyanin, rusticyanin,pseudoazurin, auracyanin or azurin-like protein, or to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 29, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36and SEQ ID NO: 43. In some embodiments, the peptide is derived fromPseudomonas aeruginosa, Phormidium laminosum, Thiobacillus ferrooxidans,Achromobacter cycloclastes, Pseudomonas syringa, Neisseria meningitidis,Vibrio parahaemolyticus, Bordetella bronchiseptica, Bordetellapertussis, Chloroflexus aurantiacus and Neisseria gonorrhoeae. In otherembodiments, the peptide is at least 10 residues and not more than 50residues in length. In some embodiments, the peptide comprises asequence which has at least about 90% amino acid sequence identity toSEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ IDNO: 47. In other embodiments, the peptide comprises or consists of SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 37, SEQID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO:47. In some embodiments, the peptide comprises the amino acid sequenceDGXXXXXDXXYXKXXD (SEQ ID NO: 35) and DGXXXXDXXYXKXXD (SEQ ID NO: 48),where D is aspartic acid, G is glycine, Y is tyrosine, K is lysine and Xis any amino acid. Finally, in some embodiments, the peptide hassignificant structural homology to the 50-77 amino acid region ofPseudomonas aeruginosa azurin.

Another aspect of the invention is a complex comprising a cargo compoundand an amino acid sequence, where the amino acid sequence has at leastabout 90% sequence identity with a cupredoxin, or a fragment thereof,the amino acid sequence, or fragment thereof, is linked to the cargocompound, and the amino acid sequence facilitates entry of the cargocompound into a mammalian cancer cell. In some embodiments, the aminoacid sequence of this complex has at least about 90% amino acid sequenceidentity to less than a full length wild-type cupredoxin or H.8 outermembrane protein. In other embodiments, the cargo compound is protein,lipoprotein, polypeptide, peptide, polysaccharide, nucleic acid, dye,microparticle, nanoparticle, toxin or drug. In particular embodimentsthe cargo is a protein or polypeptide which is linked amino acidsequence to form a fusion protein. In other particular embodiments, thecargo compound is a toxin, more particularly, the Pseudomonas aeruginosaexotoxin A. In other embodiments, the cargo is a detectable substance,more specifically one detectable by fluorimetry, microscopy, X-ray CT,MRI or ultrasound. Finally, the invention also encompasses the complexin a pharmaceutically suitable carrier.

Another aspect of the present invention is directed to a method fordelivering a cargo compound into a cell. In one embodiment, this methodcomprises contacting a cell or cells with the above complex. In otherembodiments, the cell or cells originate from a patient suffering fromcancer, and are reintroduced into the patient. In other embodiments, thecell is a cancer cell, more specifically an osteosarcoma cell, lungcarcinoma cell, colon carcinoma cell, lymphoma cell, leukemia cell, softtissue sarcoma cell, breast carcinoma cell, liver carcinoma cell,bladder carcinoma cell or prostate carcinoma cell. In other embodiments,the complex is administered to a patient in a therapeutically effectiveamount. In other embodiments, the complex is administered intravenously,topically, subcutaneously, intramuscularly or into a tumor. In otherembodiments, the complex is co-administered with another cancertreatment.

Another aspect of the invention is a method to diagnose cancer. In someembodiments, the complex with a cargo that is a detectable substance isadministered to a patient with cancer and the location of the cargo isdetected. In particular embodiments, the cargo compound is an X-raycontrast agent and is detected by X-ray CT, the cargo compound is amagnetic resonance imaging contrast agent and is detected by MRI, or thecargo is an ultrasound contrast agent and is detectable by ultrasound.In other embodiments, the cell or cells are contacted with a complexwith a detectable substance and the location of the cargo is detected.

Another aspect of the invention is a kit that contains any of the abovecomplexes. In some embodiments, the kit further comprises apharmaceutically acceptable adjuvant or excipient. In other embodiments,the kit further comprises a vehicle for administration of the reagent.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of wt-azurin from Pseudomonasaeruginosa.

SEQ ID NO: 2 is the amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 3 is the amino acid sequence of rusticyanin from Thiobacillusferrooxidans.

SEQ ID NO: 4 is the amino acid sequence of pseudoazurin fromAchromobacter cycloclastes.

SEQ ID NO: 5 is the amino acid sequence of the 36-128 amino acidfragment of wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 6 is the amino acid sequence of the 36-89 amino acid fragmentof wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 7 is the amino acid sequence of the 36-77 amino acid fragmentof wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 8 is the amino acid sequence of the 36-50 amino acid fragmentof wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 9 is the amino acid sequence of the 50-77 amino acid fragmentof wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 10 is the amino acid sequence of the 50-66 amino acidfragment of wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 11 is the amino acid sequence of the azu 67-77 amino acidfragment of wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 12 is the amino acid sequence of the forward primer forpGST-azu 36-128.

SEQ ID NO: 13 is the amino acid sequence of the reverse primer forpGST-azu 36-128.

SEQ ID NO: 14 is the amino acid sequence of the forward primer forpGST-azu 36-50.

SEQ ID NO: 15 is the amino acid sequence of the reverse primer forpGST-azu 36-50.

SEQ ID NO: 16 is the amino acid sequence of the forward primer forpGST-azu 36-77.

SEQ ID NO: 17 is the amino acid sequence of the reverse primer forpGST-azu 36-77.

SEQ ID NO: 18 is the amino acid sequence of the forward primer forpGST-azu 36-89.

SEQ ID NO: 19 is the amino acid sequence of the reverse primer forpGST-azu 36-89.

SEQ ID NO: 20 is the amino acid sequence of the forward primer forpGST-azu 50-77.

SEQ ID NO: 21 is the amino acid sequence of the forward primer forpGST-azu 67-77.

SEQ ID NO: 22 is the amino acid sequence of the reverse primer forpGST-azu 50-77 and pGST-azu 67-77.

SEQ ID NO: 23 is the amino acid sequence of the forward primer forpGST-azu 50-66.

SEQ ID NO: 24 is the amino acid sequence of the reverse primer forpGST-azu 50-66.

SEQ ID NO: 25 is the amino acid sequence of the forward primer for thegreen fluorescent protein gene.

SEQ ID NO: 26 is the amino acid sequence of the reverse primer for greenfluorescent protein gene.

SEQ ID NO: 27 is the amino acid sequence of the forward primer forgst-gfp-azu 50-77.

SEQ ID NO: 28 is the amino acid sequence of the reverse primer forgst-gfp-azu 50-77.

SEQ ID NO: 29 is the amino acid sequence of azurin from Pseudomonassyringae.

SEQ ID NO: 30 is the amino acid sequence of azurin/H.8 outer membraneprotein from Neisseria meningitides.

SEQ ID NO: 31 is the amino acid sequence of the azurin from Vibrioparahaemolyticus.

SEQ ID NO: 32 is the amino acid sequence of the azurin from Bordetellabronchiseptica.

SEQ ID NO: 33 is the amino acid sequence of the auracyanin A fromChloroflexus aurantiacus

SEQ ID NO: 34 is the amino acid sequence of the auracyanin B fromChloroflexus aurantiacus.

SEQ ID NO: 35 is an artificial amino acid sequence representing theconserved residues in the cupredoxin entry domain where D is asparticacid, G is glycine, Y is tyrosine, K is lysine and X is any amino acid.

SEQ ID NO: 36 is the amino acid sequence of the Neisseria gonorrhoeaeLaz protein.

SEQ ID NO: 37 is the amino acid sequence of the 50-67 amino acidfragment of wt-azurin from Pseudomonas aeruginosa.

SEQ ID NO: 38 is the amino acid sequence of the 57-89 amino acidfragment of auracyanin B of Chloroflexus aurantiacus.

SEQ ID NO: 39 is the amino acid sequence of the 50-77 amino acidfragment of azurin from Bordetella pertussis.

SEQ ID NO: 40 is the amino acid sequence of the 106-132 amino acidfragment of the Laz protein from Neisseria meningitidis.

SEQ ID NO: 41 is the amino acid sequence of the 53-70 amino acidfragment of azurin from P. aeruginosa.

SEQ ID NO: 42 is the amino acid sequence of the 53-64 amino acidfragment of azurin from P. aeruginosa.

SEQ ID NO: 43 is the amino acid sequence of the azurin from Bordetellapertussis.

SEQ ID NO: 44 is the amino acid sequence of the 51-77 amino acidfragment from azurin from P. aeruginosa.

SEQ ID NO: 45 is the amino acid sequence of the 51-77 amino acidfragment from azurin from Pseudomonas syringae.

SEQ ID NO: 46 is the amino acid sequence of the is the 52-78 amino acidfragment from azurin from Vibrio parahaemolyticus.

SEQ ID NO: 47 is the amino acid sequence of the 51-77 amino acidfragment from azurin from Bordetella bronchiseptica.

SEQ ID NO: 48 is an artificial amino acid sequence representing theconserved residues in the cupredoxin entry domain where D is asparticacid, G is glycine, Y is tyrosine, K is lysine and X is any amino acid.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1( a) and (b). Graphs showing that entry of azurin correlates withcytotoxicity. (a) MTT assays were performed for the determination ofwt-azurin-induced cytotoxicity against J774, UISOMel-2 and fibroblastcells. (b) Analysis of cell cycle progression in human normal fibroblastcells treated with M44KM64E mutant azurin. Fibroblast cells wereincubated with 0 (control), 0.5 or 1.0 mg/ml of mutant azurin for 24 hr.At the end of the treatment, DNA content in the cells was determined byflow cytometry.

FIGS. 2 (a) and (b). (a) Schematic representation of various truncatedazurin constructs and their purification profiles. Various fragments ofazu gene were fused at the 3′-end of the gst gene in frame. (b) GST-azufusion proteins were purified after cellular growth and lysis, loaded onSDS-PAGE and visualized by Coomassie Blue staining.

FIGS. 3 (a), (b) and (c). (a) Diagram showing construction of aGST-GFP-azu 50-77 fusion protein. The gfp gene was introduced at the3′-end of the gst gene (for GST-GFP) and the azu 50-77 fragment was thenligated at the 3′-end of the gfp gene in frame to produce theGST-GFP-azu 50-77 fusion protein. GST-GFP-azu 50-77 was purified as asingle fusion protein from the cell lysates. Purified proteins were runon SDS-PAGE and detected by Coomassie Blue staining (9(b)) and also byWestern blotting using anti-azurin antibody (9(c)).

FIGS. 4 (a), (b) and (c). Diagrams showing a kinetic study for theinternalization of GST-Green Fluorescent Protein (GFP) andGST-GFP-azurin fusion proteins. Green fluorescence was assayed in J774cells treated with various concentrations of GST-GFP (10(a)) orGST-GFP-azu 50-77 (10(b)) at 37° C. for 1 hr. Ten thousand cells wereanalyzed by flow cytometry. (c) Time-dependence of internalization ofGST-GFP-azu 50-77. J774 cells were incubated with 200 μg/ml GST-GFP-azu50-77 for indicated times at 37° C. and analyzed by flow cytometry.

FIGS. 5 (a), (b) and (c). (a) Diagram showing the exotoxin A domain III(amino acids 405-613), as well as part of domain 1b (amino acids381-404), fused to GST (GST-PEDIII) as earlier described for the GST-GFPfusion. The azu 50-77 fragment was then ligated to the carboxyl end ofGST-PEDIII (GST-PEDIII-azu 50-77), using PCR. (b) The fusion proteinswere purified by glutathione Sepharose 4B column gel filtration columnchromatography and run on SDS-PAGE for size determination. (c) Diagramshowing action of GST-PEDIII-azu 50-77 fusion protein in UISO-Mel-2cancer cells and in normal fibroblast (FBT) cells, as determined byPEDIII-mediated cytotoxicity. Various concentrations, as indicated, ofGST-PEDIII and GST-PEDIII-azu 50-77 were incubated with UISO-Mel-2 andFBT cells for 24 h, after which the cell viability was determined by MTTassay.

FIG. 6. Diagram showing the localization of the α-helix in wt-azurin aswell as in the wt-azurin 50-77 protein transduction domain. Replacementof three amino acids in the azurin 50-77 domain by proline residues isindicated.

FIG. 7. Diagram PEDIII-mediated cytotoxicity of GST-PEDIII-rusticyaninfusion protein against UISO-Mel-2 cancer cells and FBT cells. Variousconcentrations, as indicated, of GST-PEDIII and GST-PEDIII-azu 50-77were incubated with UISO-Mel-2 and FBT cells for 24 h, after which thecell viability was determined by MTT assay.

FIGS. 8 (a) and (b). (a) Diagram showing the structural alignment ofazurin with other cupredoxins, as computed by the VAST algorithm. TheN-terminal extended parts of auracyanin B and rusticyanin, being absentin azurin, have been omitted in the figure. The gray bars indicate theregions of azurin that can be superimposed on residues from eachneighbor. The blank spaces are unaligned regions. The azurin proteintransduction domain (PTD), azurin amino acids 50-77, where there is noalignment with rusticyanin is highlighted by vertical dashed lines. Thenumbers in parentheses after the names of the cupredoxins are theprotein database accession numbers. (b). Multiple amino acid sequencealignment of the residues comprising the middle part in some knownbacterial azurins. Bacterial genus and species are abbreviated asfollows: Psae, Pseudomonas aeruginosa (SEQ ID NO:44); Pssy, Pseudomonassyringae (SEQ ID NO: 45); Neme, Neisseria meningitidis (SEQ ID NO:40);Vipa, Vibrio parahaemolyticus (SEQ ID NO:46); Bobr, Bordetellabronchiseptica (SEQ ID NO:47). Numbers of intervening amino acids aregiven in parenthesis. The CLUSTAL X software (Higgins and Sharp, Gene101:6427-6432 (1988)) was used to generate this multiple sequencealignment.

FIG. 9. FIG. 9 depicts a diagram of the Laz protein from Neisseriameningitidis and the azurin protein from Pseudomonas aeruginosa. Thewording to the left of the bar indicates the name of the protein. Thewording above the bars indicate the name of the region of the proteindirectly below in the bar. The numbers below the bar indicate the aminoacid number of the junction of the regions of the protein.

FIG. 10. FIG. 10 depicts the constructs of several azurin fusionproteins. The wording to left of the bar indicates the name of theconstruct, with plasmid and protein product indicated. The wording abovethe bar indicates the region of the protein depicted directly below inthe bar. “N.Sp” indicates the Neisseria gonorrhoeae signal peptide;“H.8” indicates the H.8 region of Neisseria gonorrhoeae; “N.Azu”indicates Neisseria gonorrhoeae azurin; “P.Sp” indicates the Pseudomonasaeruginosa signal peptide; and “P.Azu” indicates the P. aeruginosasignal peptide.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention relates to methods and materials for delivering acargo compound into a cell. Delivery of the cargo compound according tothis invention is accomplished by the use of a suitable transportpolypeptide. In one embodiment of the invention, the cargo compound islinked to the transport polypeptide. Suitable transport peptides includea cupredoxin, or a fragment of a cupredoxin containing a “cupredoxinentry domain”. The term “cupredoxin entry domain” refers to a fragmentof a cupredoxin that includes the amino sequence that is required forthe entry of cupredoxin into a mammalian cancer cell. Cargo compoundsdelivered by the present invention include, but are not limited to,proteins, lipoproteins, polypeptides, peptides, polysaccharides, nucleicacids, including anti-sense nucleic acids, dyes, fluorescent andradioactive tags, microparticles or nanoparticles, toxins, inorganic andorganic molecules, small molecules, and drugs. In some embodiments, thedrugs and toxins kill tumor cells.

In one embodiment of the invention, the cupredoxin is an azurin, such aswt-azurin from Pseudomonas aeruginosa. “Wt-azurin” refers to wild-typeazurin from P. aeruginosa. Similarly, the term “wt-azurin entry domain”refers to a fragment of wt-azurin that includes the amino sequence thatis required for the entry of wt-azurin into a cell. In other embodimentsof the invention, the cupredoxin is a plastocyanin, a rusticyanin, or apseudoazurin, among others. In specific embodiments, the azurin is fromPseudomonas aeruginosa, Pseudomonas syringa, Neisseria meningitides,Neisseria gonorrhoeae, Vibrio parahaemolyticus or Bordetellabronchiseptica, among others.

In one embodiment, a cargo compound is delivered to kill or retard cellcycle progression in a cell, such as a cancer cell. Such a cancer cellcan be, for example, an osteosarcoma cell, lung carcinoma cell, coloncarcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcoma cellor breast, liver, bladder or prostate carcinoma cell, among others. Forexample, the cargo compound can be a cell cycle control protein, such asp53; a cyclin-dependent kinase inhibitor, such as p16, p21 or p27; asuicide protein such as thymidine kinase or nitroreductase; a cytokineor other immunomodulatory protein such as interleukin 1, interleukin 2or granulocyte-macrophage colony stimulating factor (GM-CSF); or atoxin, such as Pseudomonas aeruginosa exotoxin A, among others. In otherembodiments, a biologically active fragment of one of the above classesof compounds is delivered. In another embodiment, the cargo compound isdelivered in order to generate an image of the target tissue. Forexample, the target tissue can be a cancer and the cargo compound can beone commonly used to generate an image for detection by X-ray computedtomography (CT), Magnetic Resonance Imaging (MRI) and ultrasound. Inthese embodiments, the cargo compound is a gamma ray or positronemitting radioisotope, a magnetic resonance imaging contrast agent, anX-ray contrast agent, or an ultrasound contrast agent.

Cupredoxins

“Cupredoxins” are small blue copper containing proteins having electrontransfer properties (10-20 kDa) that participate in, for example,bacterial redox chains or photosynthesis. The copper ion is solely boundby the protein matrix. A special distorted trigonal planar arrangementto two histidine and one cysteinate ligands around the copper gives riseto very peculiar electronic properties of the metal site and an intenseblue color. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution. The cupredoxins include theazurins, plastocyanins, rusticyanins, pseudoazurins, auracyanins andazurin-like proteins. As used herein, the term “cupredoxin” includes theprotein form without the copper atom present, as well as the coppercontaining protein.

Azurins

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin plants and certain bacteria. The azurins include those from P.aeruginosa (SEQ ID NO: 1) (“wt-azurin”), A. xylosoxidans, and A.denitrificans. Murphy, L. M. et al., J. Mol. Biol. 315:859-71 (2002).Although the sequence homology between the azurins varies between60-90%, the structural homology between these molecules is high. Allazurins have a characteristic β-sandwich with Greek key motif and thesingle copper atom is always placed at the same region of the protein.In addition, azurins possess an essentially neutral hydrophobic patchsurrounding the copper site (Murphy et al.).

Plastocyanins

The plastocyanins are cupredoxins that are found in eukaryotic plantsand cyanobacteria. They contain one molecule of copper per molecule andare blue in their oxidized form. They occur in the chloroplast, wherethey function as electron carriers. Since the determination of thestructure of poplar plastocyanin in 1978, the structure of algal(Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean)plastocyanins has been determined either by crystallographic or NMRmethods, and the poplar structure has been refined to 1.33 Å resolution.SEQ ID NO: 2 shows the amino acid sequence of plastocyanin from thecyanobacterium Phormidium laminosum.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths in plastocyanin. One putative electron transfer path is relativelyshort (approximately 4 Å) and involves the solvent-exposed copper ligandHis-87 in the hydrophobic patch, while the other is more lengthy(approximately 12-15 Å) and involves the nearly conserved residue Tyr-83in the negative patch. Redinbo et al., J. Bioenerg. Biomembr.26(1):49-66 (1994).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a thiobacillus. The X-ray crystal structure of theoxidized form of the extremely stable and highly oxidizing cupredoxinrusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 3) has beendetermined by multiwavelength anomalous diffraction and refined to 1.9 Åresolution. The rusticyanins are composed of a core beta-sandwich foldcomposed of a six- and a seven-stranded β-sheet. Like other cupredoxins,the copper ion is coordinated by a cluster of four conserved residues(His 85, Cys138, His143, Met148) arranged in a distorted tetrahedron.Walter, R. L. et al., J. Mol. Biol. 263:730-51 (1996).

Auracyanins

Three small blue copper proteins designated auracyanin A, auracyanin B1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms have almost identical properties to each other, but are distinctfrom the A form. The sodium dodecyl sulfate-polyacrylamide gelelectrophoresis demonstrates apparent monomer molecular masses as 14(A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. (Van Dreissche et al,Protein Science 8:947-957 (1999). His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 33 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. (Bond et al., J. Mol. Biol. 306:47-67 (2001).) With theexception of an additional N-terminal strand, the molecule is verysimilar to that of the bacterial cupredoxin, azurin. As in othercupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide,and the other three lie along a large loop between strands 7 and 8. TheCu site geometry is discussed with reference to the amino acid spacingbetween the latter three ligands. The crystallographically characterizedCu-binding domain of auracyanin B is probably tethered to theperiplasmic side of the cytoplasmic membrane by an N-terminal tail thatexhibits significant sequence identity with known tethers in severalother membrane-associated electron-transfer proteins. The amino acidsequences of the B forms are presented in McManus et al. (J Biol Chem.267:6531-6540 (1992).). See exemplary amino acid sequence SEQ ID NO: 34for chain A of auracyanin B from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptides. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 4. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Cytotoxic Activity of Cupredoxins

Cupredoxins have been studied extensively for their electron transfer(redox) properties but until recently were not known to exhibitcytotoxic effects. The present invention is predicated by the inventors'surprising discovery that cupredoxins as well as the iron (haem)containing redox protein cytochrome c551 induce either apoptosis orinhibit cell cycle progression in J774 mouse macrophage tumor cells andin human cancer cells. The redox activity of cupredoxins is not criticalfor their cytotoxic activity. For example, cupredoxins without a copperatom often exhibit a much lower redox activity compared to thosecontaining the copper atom, but nevertheless demonstrate significantcytotoxic activity. In comparison to their activity in cancer cells,cupredoxins induce only a low level of apoptosis in vivo in normaltissues of tumor-bearing cupredoxin-treated mice.

The cytotoxic activity of cupredoxins is described in co-pending U.S.patent application Ser. No. 10/047,710, filed Jan. 15, 2002, and inco-pending U.S. patent application Ser. No. 10/720,603, filed Nov. 24,2003. These prior applications are hereby incorporated by reference.

The present inventors now show that the selective effect of cupredoxinson cancer cells is related to the ability of cupredoxins to enter thesecells. In Example 5, the inventors show that cupredoxins enter J774cells. These cells are ascites forms of murine reticulum cell sarcomawith macrophage-like properties. In Examples 18 and 19, the inventorshave shown that an azurin-like protein, the H.8 outer membrane fromNeisseria, also know as Laz, can specifically enter brain tumor cells.In comparison, cupredoxins show an extremely reduced rate of entry intonormal cells.

In one embodiment, the present invention relates to a “complex”containing a cupredoxin, or a fragment of a cupredoxin, linked to a“cargo compound” that is to be delivered into a cell. The cargo compoundmay be linked either covalently or non-covalently to form the complex.Methods of preparing such a complex are well known to those skilled inthe art. For example, if the cargo compound is a protein or polypeptide,the complex can be formed as a fusion protein. Alternatively, the cargocompound may be covalently linked to the cupredoxin, or cupredoxinfragment, either directly or through a linker molecule, via, for examplea disulfide or ester linkage.

Cupredoxin Entry Domain

The invention provides for a protein transduction domain that allows forthe transport of linked cargo into mammalian cancer cells but notnon-cancerous cells. It has been discovered that cupredoxin proteinscomprise a protein transduction domain, the cupredoxin entry domain,which facilitates the entry of linked cargo into mammalian cancer cells.In some embodiments, the entire cupredoxin protein can be used tofacilitate the transport linked cargo selectively into cancer cells. Inother embodiments, a portion of a cupredoxin can be used to transportlinked cargo into cancer cells. In some embodiments, the cupredoxinentry domain consists of a region of a cupredoxin that is less than thefull length wild-type protein. In some embodiments, the cupredoxin entrydomain consists of more than about 10 residues, about 15 residues orabout 20 residues of a cupredoxin. In some embodiments, the cupredoxinentry domain consists of not more than about 50 residues, about 40residues or about 30 residues of a cupredoxin. In some embodiments, thecupredoxin entry domain has at least about 90% amino acid sequenceidentity, at least about 95% amino acid sequence identity or at leastabout 99% amino acid sequence identity to a cupredoxin.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. A“polypeptide”, “peptide” or “protein” may be synthesized within a celland isolated from other proteins and cellular components. Alternatively,a “polypeptide”, “peptide” or “protein” may be artificially synthesizedaccording to methods well known to those in the art, and thus also befree of other proteins. The terms apply to amino acid polymers in whichone or more amino acid residue is an artificial chemical analogue of acorresponding naturally occurring amino acid. The terms also apply tonaturally occurring amino acid polymers. The terms “polypeptide,”“peptide,” and “protein” are also inclusive of modifications including,but not limited to, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation. It will be appreciated that polypeptides are notalways entirely linear. For instance, polypeptides may be branched as aresult of ubiquitination and they may be circular (with or withoutbranching), generally as a result of post-translation events, includingnatural processing event and events brought about by human manipulationwhich do not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods as well.

The Examples describe a method of identifying fragments of P. aeruginosawt-azurin that are suitable for use in the present invention. Such amethod can also be used to identify fragments of other cupredoxins.Examples 1 and 2 describe the construction of a series of glutathioneS-transferase (“GST”) fusions of wt-azurin truncated at both the N- andthe C-terminal. These examples also describe the purification of thefusion protein products.

Example 9 shows the internalization of such fusions in J774 cells at 37°C. While wt-azurin was internalized, GST remained at the periphery ofthe cells and was not internalized. azu 36-128 (SEQ ID NO: 5) and azu36-89 (SEQ ID NO: 6) were internalized, as was azu 36-77 (SEQ ID NO: 7).Further truncations show that, while azu 50-77 (SEQ ID NO: 9) isinternalized, the internalization of azu 36-50 (SEQ ID NO: 8) is highlyinefficient. Further truncations of azu 50-77 (SEQ ID NO: 9) to azu50-66 (SEQ ID NO: 10) and azu 67-77 (SEQ ID NO: 11) demonstrate verylittle internalization, indicating that efficient internalizationrequires not interfering with the sequence at or about positions 66-67.From a practical standpoint, the data support the use of amino acids 50to 77 for efficient transport.

In some embodiments, the cupredoxin entry domain is a wt-azurin entrydomain. In one embodiment of the present invention, a wt-azurin entrydomain contains at least amino acids 50 to 77 of wt-azurin (SEQ ID NO:9). In another embodiment of the invention, the wt-azurin entry domaincontains at least amino acids 36 to 77 of wt-azurin (SEQ ID NO: 7). Inanother embodiment of the invention, the wt-azurin entry domain containsat least amino acids 36 to 89 of wt-azurin (SEQ ID NO: 6). In anotherembodiment of the invention, the wt-azurin entry domain contains atleast amino acids 36 to 128 of wt-azurin (SEQ ID NO: 5). In yet anotherembodiment of the invention, the wt-azurin entry domain contains atleast amino acids 50 to 67 of wt-azurin (SEQ ID NO: 37). In anotherembodiment of the invention, the wt-azurin entry domain contains atleast amino acids 53 to 70 of wt-azurin (SEQ ID NO: 41). In yet anotherembodiment of the invention, the wt-azurin entry domain contains atleast amino acids 53 to 64 of wt-azurin (SEQ ID NO: 42).

In another embodiment of the invention, the cupredoxin entry domain isan entry domain from a cupredoxin other than P. aeruginosa azurin. Indifferent embodiments, the cupredoxin entry domain may be a fragment ofplastocyanin from the cyanobacterium Phormidium laminosum (SEQ ID NO:2), rusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 3);pseudoazurin from Achromobacter cycloclastes (SEQ ID NO: 4), azurin fromPseudomonas syringae (SEQ ID NO: 29), azurin from Neisseria meningitidis(SEQ ID NO: 30), azurin from Neisseria gonnorhoeae (SEQ ID NO: 36),azurin from Vibrio parahaemolyticus (SEQ ID NO: 31), azurin fromBordetella bronchiseptica (SEQ ID NO: 32), azurin from Bordetellapertussis (SEQ ID NO: 43) or an auracyanin from Chloroflexus aurantiacus(SEQ ID NO: 33 and 34).

In another embodiment of the invention, the cupredoxin entry domaincontains at least amino acids 57 to 89 of auracyanin B of Chloroflexusaurantiacus (SEQ ID NO: 38). In another embodiment of the invention, thecupredoxin entry domain contains at least amino acids 50 to 77 ofBordetella pertussis (SEQ ID NO: 39). In another embodiment of theinvention, the cupredoxin entry domain contains at least amino acids 106to 132 of N. meningitidis (SEQ ID NO: 40). In another embodiment of theinvention, the cupredoxin entry domain contains at least amino acids51-77 of Pseudomonas syringae azurin (SEQ ID NO: 45). In anotherembodiment of the invention, the cupredoxin entry domain contains atleast amino acids 89-115 of Neisseria meningitidis Laz (SEQ ID NO: 40).In another embodiment of the invention, the cupredoxin entry domaincontains at least amino acids 52-78 of Vibrio parahaemolyticus azurin(SEQ ID NO: 46). In another embodiment of the invention, the cupredoxinentry domain contains at least amino acids 51-77 of Bordetellabronchiseptica azurin (SEQ ID NO: 47).

Modification of a Cupredoxin Entry Domain

In another embodiment of the present invention, a cupredoxin entrydomain is chemically modified or genetically altered to produce variantsthat retain the ability to transport a cargo compound into a cell. Forexample, Example 14 shows that wt-azurin having proline residuesintroduced at positions 54, 61 and 70 retains its ability to enterUISO-Mel-2 cells.

In another embodiment, the cupredoxin entry domain comprises a conservedamino acid sequence DGXXXXXDXXYXKXXD (SEQ ID NO: 35) or DGXXXXDXXYXKXXD(SEQ ID NO: 48) where D is aspartic acid, G is glycine, Y is tyrosine, Kis lysine and X is any amino acid. See Example 17.

Variants of a cupredoxin entry domain may be synthesized by standardtechniques. Derivatives are amino acid sequences formed from nativecompounds either directly or by modification or partial substitution.Analogs are amino acid sequences that have a structure similar, but notidentical, to the native compound but differ from it in respect tocertain components or side chains. Analogs may be synthesized or from adifferent evolutionary origin.

Variants may be full length or other than full length, if the derivativeor analog contains a modified amino acid. Variants of a cupredoxin entrydomain include, but are not limited to, molecules comprising regionsthat are substantially homologous to the cupredoxin entry domain by atleast about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% identity over anamino acid sequence of identical size or when compared to an alignedsequence in which the alignment is performed by a homology algorithm.

The term “percent (%) amino acid sequence identity” between a cupredoxinentry domain and a candidate sequence is defined as the percentage ofamino acid residues in a cupredoxin entry domain that are identical withamino acid residues in a candidate sequence when the two sequences arealigned. To determine % amino acid identity, sequences are aligned andif necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y·100where

-   -   X is the number of amino acid residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B    -   and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A.

Changes can be introduced into a cupredoxin entry domain that incuralterations in the amino acid sequences of the cupredoxin entry domainthat do nullify the ability of the cupredoxin entry domain to transporta cargo compound into a cell. A “non-essential” amino acid residue is aresidue that can be altered from the sequence of the cupredoxin entrydomain without nullifying its ability to transport a cargo compound intoa cell, whereas an “essential” amino acid residue is required for suchactivity.

Amino acids for which “conservative” substitutions can be made are wellknown in the art. Useful conservative substitutions are shown in Table1, “Preferred substitutions.” Conservative substitutions whereby anamino acid of one class is replaced with another amino acid of the sameclass fall within the scope of the invention so long as the substitutiondoes not nullify the activity of the cupredoxin entry domain. Suchexchanges that result in altered cupredoxin entry domain activity arecontemplated as part of the invention so long as such activity isappreciable.

TABLE 1 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L)Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norleucine Leu

“Non-conservative” substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the cupredoxin entry domain function. Residuesare divided into groups based on common side-chain properties as denotedin Table 2. Non-conservative substitutions entail exchanging a member ofone of these classes for another class.

Non-conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of a different class fall within thescope of the invention so long as the substitution does not nullify theactivity of the cupredoxin entry domain. Such exchanges that result inaltered cupredoxin domain activity are contemplated as part of theinvention so long as such activity is appreciable.

TABLE 2 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

In another embodiment, the variants of a cupredoxin entry domain have asignificant structural similarity to P. aeruginosa azurin residues50-77. Examples of studies that determine significant structuralhomology between cupredoxins and other proteins include Toth et al.(Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a variant of the cupredoxin entry domain andP. aeruginosa azurin residues 50-77 is determined by using the VASTalgorithm (Gibrat et al., Curr Opin Struct Biol 6:377-385 (1996); Madejet al., Proteins 23:356-3690 (1995)). In specific embodiments, the VASTp value from a structural comparison of a variant of the cupredoxinentry domain and P. aeruginosa azurin residues 50-77 is less than about10⁻³, less than about 10⁻⁵, or less than about 10⁻⁷. In otherembodiments, significant structural homology between a variant of thecupredoxin entry domain and P. aeruginosa azurin residues 50-77 can bedetermined by using the DALI algorithm (Holm & Sander, J. Mol. Biol.233:123-138 (1993)). In specific embodiments, the DALI Z score for apairwise structural comparison is at least about 3.5, at least about7.0, or at least about 10.0.

Modifications to the cupredoxin entry domain can be made using methodsknown in the art such as oligonucleotide-mediated (site-directed)mutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis (Carter, Biochem J. 237:1-7 (1986); Zoller and Smith,Methods Enzymol. 154:329-50 (1987)), cassette mutagenesis, restrictionselection mutagenesis (Wells et al., Gene 34:315-23 (1985)) or otherknown techniques can be performed on the cloned DNA to produce acupredoxin entry domain variant nucleic acid. In addition, nucleotidesencoding entry domains with structural similarity to that of thecupredoxin entry domains may be synthesized by methods that are wellknown in the art. Further, protein molecules that are wild type orvariant cupredoxin entry domains may be synthesized by methods that arewell known in the art.

Nucleic Acids Coding for a the Cupredoxin Entry Domain and Complex of aCupredoxin Entry Domain Linked to a Cargo Compound

In another aspect, the present invention provides a nucleic acidmolecule encoding a fusion protein comprising a cupredoxin entry domainlinked to a cargo compound, where the cargo compound is a protein orpeptide. The nucleic acid molecule according to the invention can beprepared by a combination of known techniques in the art. For instance,nucleic acid sequences for the cupredoxin entry domain and the cargocompound can individually be prepared by chemical synthesis or cloning.The nucleic acid sequences are then ligated in order with a ligase togive a nucleic acid molecule of interest.

Methods of Delivering a Cargo Compound using a Cupredoxin Entry Domain

Many arginine-rich peptides are known to translocate through mammaliancell membranes and carry protein cargo compounds inside such cells.Suzuki, T., et al. J. Biol. Chem. 277:2437-43 (2002). For example, ashort arginine-rich 11 amino acid (amino acids 47-57) segment of HIV Tatprotein allows transport of cargo proteins into mammalian cells.Schwarze, S R., et al. Trends Cell Biol. 10:290-95 (2000). Syntheticentry domains that strengthen the alpha-helical content and optimize theplacement of arginine residues have been shown to have enhancedpotential as protein transduction domains. Ho, A., et al. Cancer Res.61:474-77 (2001). In comparison, wt-azurin has a single arginineresidue. It is therefore believed, but not relied upon for the presentinvention, that its mode of entry is different from that of the Tatprotein.

The present invention encompasses the use of those cupredoxin fragmentsthat facilitate the entry of a cargo compound into a cell. Suchfragments may be determined by any method that identifies thosefragments required for entry into a cell. In one such method, acupredoxin fragment is linked to a marker substance and a test performedto determine whether the cupredoxin fragment enters a cell. Such methodsmay be used to identify suitable fragments of the cupredoxins discussedabove.

In various embodiments of the present invention, the cargo compound isattached to a cupredoxin, such as azurin from P. aeruginosa (SEQ IDNO: 1) (“wt-azurin”); plastocyanin from the cyanobacterium Phormidiumlaminosum (SEQ ID NO: 2); rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 3); or pseudoazurin from Achromobacter cycloclastes (SEQ IDNO: 4), azurins from Pseudomonas syringa (SEQ ID NO: 29), Neisseriameningitidis (SEQ ID NO: 30), Vibrio parahaemolyticus (SEQ ID NO: 31),Bordetella bronchiseptica (SEQ ID NO: 32), auracyanin A and B fromChloroflexus aurantiacus (SEQ ID NO. 33 and 34) or Neisseria gonorrhoeae(SEQ ID NO. 36), among other azurin and azurin-like proteins. In otherembodiments, the cargo is linked to a cupredoxin entry domain.

In various embodiments of the present invention, a cupredoxin entrydomain delivers a cargo compound into a cell in vitro, ex vivo or invivo. For example, delivery may be achieved in vitro by adding a complexof a cupredoxin entry domain and a cargo compound to a cell culture,such as a pap smear. Alternatively, delivery may be achieved ex vivo byadding the complex to a sample removed from a patient, for example,blood, tissue, or bone marrow, and returning the treated sample to thepatient. Delivery may also be achieved by administration of the complexdirectly to a patient. The methods of the present invention may be usedfor therapeutic, prophylactic, diagnostic or research purposes. Cargocompounds delivered by the present invention include, but are notlimited to, proteins, lipoproteins, polypeptides, peptides,polysaccharides, nucleic acids, including anti-sense nucleic acids,dyes, microparticles or nanoparticles, toxins, organic and inorganicmolecules, small molecules, and drugs.

In one embodiment, a detectable substance, for example, a fluorescentsubstance, such as green fluorescent protein; a luminescent substance;an enzyme, such as β-galactosidase; or a radiolabelled or biotinylatedprotein is delivered to confer a detectable phenotype to a cell.Similarly, microparticles or nanoparticles labeled with a detectablesubstance, for example, a fluorescent substance, can be delivered. Oneexample of suitable nanoparticles is found in U.S. Pat. No. 6,383,500,issued May 7, 2002, which is hereby expressly incorporated by reference.Many such detectable substances are known to those skilled in the art.

In some embodiments, the cargo compound is a detectable substance thatis suitable for X-ray computed tomography, magnetic resonance imaging,ultrasound imaging or radionuclide scintigraphy. In these embodiments,the cargo compound is administered to the patient for purposes ofdiagnosis. A contrast agent is administered as a cargo compound toenhance the image obtained by X-ray CT, MRI and ultrasound. Theadministration of a radionuclide cargo compound that is targeted totumor tissue via the cupredoxin entry domain can be used forradionuclide scinitigraphy. In some embodiments, the cupredoxin entrydomain may contain the radionucleotide with or without a cargo compound.In other embodiments, the cargo compound is a gamma ray or positronemitting radioisotope, a magnetic resonance imaging contract agent, anX-ray contrast agent, or an ultrasound contrast agent.

Ultrasound contrast agents suitable for use as cargo compounds include,but are not limited to, a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein expressly incorporated by reference. The termsurfactant microsphere includes nanospheres, liposomes, vesicles and thelike. The biocompatible gas can be air, or a fluorocarbon, such as aC₃-C₅ perfluoroalkane, which provides the difference in echogenicity andthus the contrast in ultrasound imaging. The gas is encapsulated orcontained in the microsphere to which is attached the cupredoxin entrydomain, optionally via a linking group. The attachment can be covalent,ionic or by van der Waals forces. Specific examples of such contrastagents include lipid encapsulated perfluorocarbons with a plurality oftumor neovasculature receptor binding peptides, polypeptides orpeptidomimetics.

X-ray contrast agents suitable for use as cargo compounds include, butare not limited to, one or more X-ray absorbing or “heavy” atoms ofatomic number 20 or greater, further comprising an optional linkingmoiety, L_(n), between the cupredoxin entry domain and the X-rayabsorbing atoms. The frequently used heavy atom in X-ray contrast agentsis iodine. Recently, X-ray contrast agents comprised of metal chelates(e.g., U.S. Pat. No. 5,417,959) and polychelates comprised of aplurality of metal ions (e.g., U.S. Pat. No. 5,679,810) have beendisclosed. More recently, multinuclear cluster complexes have beendisclosed as X-ray contrast agents (e.g., U.S. Pat. No. 5,804,161, PCTWO91/14460, and PCT WO 92/17215).

MRI contrast agents suitable for use as cargo compounds include, but arenot limited to, one or more paramagnetic metal ions, further comprisingan optional linking moiety, L_(n), between the cupredoxin entry domainand the paramagnetic metal ions. The paramagnetic metal ions are presentin the form of metal complexes or metal oxide particles. U.S. Pat. Nos.5,412,148, and 5,760,191, describe examples of chelators forparamagnetic metal ions for use in MRI contrast agents. U.S. Pat. No.5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No. 5,281,704,describe examples of polychelants useful for complexing more than oneparamagnetic metal ion for use in MRI contrast agents. U.S. Pat. No.5,520,904, describes particulate compositions comprised of paramagneticmetal ions for use as MRI contrast agents.

In another embodiment, a cargo compound is delivered to kill or retardcell cycle progression in a cell, such as a cancer cell. Such a cancercell can be, for example, an osteosarcoma cell, lung carcinoma cell,colon carcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcomacell or breast, liver, bladder or prostate carcinoma cell. For example,the cargo compound can be a cell cycle control protein, such as p53; acyclin-dependent kinase inhibitor, such as p16, p21 or p27; a suicideprotein such as thymidine kinase or nitroreductase; a cytokine or otherimmunomodulatory protein such as interleukin 1, interleukin 2 orgranulocyte-macrophage colony stimulating factor (GM-CSF); or a toxin,such as Pseudomonas aeruginosa exotoxin A. In other embodiments, abiologically active fragment of one of the above classes of compounds isdelivered.

In yet another embodiment, the cargo compound is a nucleic acid codingfor one of the above classes of compounds. In yet another embodiment,the cargo compound is a drug used to treat cancer. Such drugs include,for example, 5-fluorouracil; Interferon α; Methotrexate; Tamoxifen; andVincristine. The above examples are provided for illustration only, manyother such compounds are known to those skilled in the art.

Cargo compounds suitable for treating cancer include, but not limitedto, alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17.beta.-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol™), docetaxel (Taxotere™), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful as cargocompounds include the following: epothilone derivatives as found inGerman Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416(see also U.S. Pat. No. 6,040,321); and prenyl-protein transferaseinhibitors as found in WO 97/30992 and WO 98/54966; and agents such asthose described generically and specifically in U.S. Pat. No. 6,011,029(the compounds of which U.S. patent can be employed together with anyNHR modulators (including, but not limited to, those of presentinvention) such as AR modulators, ER modulators, with LHRH modulators,or with surgical castration, especially in the treatment of cancer).

The above other therapeutic agents, when employed as cargo compoundswith the compounds of the present invention, may be used, for example,in those amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

Pharmaceutical Compositions Containing a Cupredoxin Entry Domain

Pharmaceutical compositions containing a complex of a cupredoxin entrydomain linked to a cargo compound can be manufactured in anyconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The complex can be readily combined with apharmaceutically acceptable carrier well-known in the art. Such carriersenable the preparation to be formulated as a tablet, pill, dragee,capsule, liquid, gel, syrup, slurry, suspension, and the like. Suitableexcipients can also include, for example, fillers and cellulosepreparations. Other excipients can include, for example, flavoringagents, coloring agents, detackifiers, thickeners, and other acceptableadditives, adjuvants, or binders.

Such compositions can be used in, for example, the detection or imagingof a cell type or in the treatment of a condition related to cell deathor in the prevention thereof. The compositions can be administered in anamount sufficient to prevent or treat a condition related to resistanceto cell death. As used herein, the term “a condition related toresistance to cell death” refers to a disease, state, or ailmentcharacterized by at least a tendency for prolonged cell life whencompared with a healthy cell of like kind as determined by a reasonable,skilled physician or clinician. Typically, the host organism is amammal, such as a human or animal.

Administration of Compositions Containing a Cupredoxin Entry Domain

Compositions containing a cupredoxin entry domain can be administered byany suitable route, for example, by oral, buccal, inhalation,sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary administration). Thecompositions and pharmaceutical formulations thereof can be administeredin any amount effective to achieve its intended purpose. Whenadministrated to treat a condition related to resistance to cell death,the composition is administered in a therapeutically effective amount. A“therapeutically effective amount” is an amount effective to preventdevelopment of, or to alleviate the existing symptoms of, the subjectbeing treated. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art.

In various embodiments, the composition includes carriers and excipients(including but not limited to buffers, carbohydrates, mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants,bacteriostats, chelating agents, suspending agents, thickening agentsand/or preservatives), water, oils, saline solutions, aqueous dextroseand glycerol solutions, other pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such asbuffering agents, tonicity adjusting agents, wetting agents and thelike. It will be recognized that, while any suitable carrier known tothose of ordinary skill in the art may be employed to administer thecompositions of this invention, the type of carrier will vary dependingon the mode of administration. Compounds may also be encapsulated withinliposomes using well-known technology. Biodegradable microspheres mayalso be employed as carriers for the compositions of this invention.Suitable biodegradable microspheres are shown, for example, in U.S. Pat.Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763,5,814,344 and 5,942,252. “Compounds” as used herein, include thepeptides, amino acid sequences, cargo compounds and complexes of thepresent invention

The half-life in the bloodstream of the compositions of the inventioncan be extended or optimized by several methods well known to those inthe art, including but not limited to, circularized peptides (Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), D,L-peptides (diastereomer), (Futaki et al., J.Biol. Chem. February 23; 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (Lee et al., J. Pept.Res. 63(2):69-84 (2004)), and N- and C-terminal modifications (Labrie etal., Clin. Invest. Med. 13(5):275-8, (1990)). Of particular interest ared-isomerization (substitution) and modification of peptide stability viaD-substitution or L-amino acid substitution.

The compositions of the invention may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

The compositions of the invention may be administered in a variety ofways, including by injection (e.g., intradermal, subcutaneous,intramuscular, intraperitoneal and the like), by inhalation, by topicaladministration, by suppository, by using a transdermal patch or bymouth.

When administration is by injection, composition may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the composition maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

When administration is by inhalation, the composition may be deliveredin the form of an aerosol spray from pressurized packs or a nebulizerwith the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the proteins and a suitable powder base suchas lactose or starch.

When administration is by topical administration, the composition may beformulated as solutions, gels, ointments, creams, suspensions, and thelike, as are well known in the art. In some embodiments, administrationis by means of a transdermal patch. When administration is bysuppository (e.g., rectal or vaginal), composition may also beformulated in compositions containing conventional suppository bases.

When administration is oral, the composition can be readily formulatedin combination with pharmaceutically acceptable carriers well known inthe art. A solid carrier, such as mannitol, lactose, magnesium stearate,and the like may be employed; such carriers enable the chemotaxin to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject tobe treated. For oral solid formulations such as, for example, powders,capsules and tablets, suitable excipients include fillers such assugars, cellulose preparation, granulating agents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include the composition allow forthe release of the composition over extended periods of time, such thatwithout the sustained release formulation, composition would be clearedfrom a subject's system, and/or degraded by, for example, proteases andsimple hydrolysis before eliciting or enhancing an therapeutic effect.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the complex which are sufficient to maintain therapeuticeffect. Generally, the desired composition is administered in anadmixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

The appropriate dosage will, of course, vary depending upon, forexample, the compound containing the cupredoxin entry domain employed,the host, the mode of administration and the nature and severity of theconditions being treated or diagnosed. However, in one embodiment of themethods of the present invention, satisfactory treatment results inhumans are indicated to be obtained at daily dosages from about 0.001 toabout 20 mg/kg of body weight of the compound containing the cupredoxinentry domain. In one embodiment, an indicated daily dosage for treatmentin humans may be in the range from about 0.7 mg to about 1400 mg of acompound containing the cupredoxin entry domain convenientlyadministered, for example, in daily doses, weekly doses, monthly doses,and/or continuous dosing. Daily doses can be in discrete dosages from 1to 12 times per day. Alternatively, doses can be administered everyother day, every third day, every fourth day, every fifth day, everysixth day, every week, and similarly in day increments up to 31 days.Dosing can be continuous, intermittent or a single dose, using anyapplicable dosing form, including tablet, patches, i.v. administrationand the like. More specifically, the composition is administered in atherapeutically effective amount. In specific embodiments, thetherapeutically effective amount is from about 0.01-20 mg/kg of bodyweight. In specific embodiments, the dose level is about 10 mg/kg/day,about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30mg/kg/day, about 35 mg/kg/day, about 40 mg/kg/day, about 45 mg/kg/day orabout 50 mg/kg/day.

The method of introducing compounds containing the cupredoxin entrydomain to patients is, in some embodiments, co-administration with otherdrugs known to treat cancer. Such methods are well-known in the art. Ina specific embodiment, the compounds containing the cupredoxin entrydomain are part of an cocktail or co-dosing containing or with otherdrugs for treating cancer. Such drugs include, for example, those listedherein and specifically 5-fluorouracil; Interferon α; Methotrexate;Tamoxifen; and Vincristine. The above examples are provided forillustration only, many other such compounds are known to those skilledin the art.

Other drugs suitable for treating cancer include, but not limited to,alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17.beta.-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol™), docetaxel (Taxotere™), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies. The compounds of the invention may also be used inconjunction with radiation therapy and surgery.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful toco-administer with the compositions of the invention include thefollowing: epothilone derivatives as found in German Patent No.4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclindependent kinase inhibitors as found in WO 99/24416 (see also U.S. Pat.No. 6,040,321); and prenyl-protein transferase inhibitors as found in WO97/30992 and WO 98/54966; and agents such as those described genericallyand specifically in U.S. Pat. No. 6,011,029 (the compounds of which U.S.patent can be employed together with any NHR modulators (including, butnot limited to, those of present invention) such as AR modulators, ERmodulators, with LHRH modulators, or with surgical techniques,especially in the treatment of cancer).

Pharmaceutical compositions used in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the composition, activeagents, for inhibiting or stimulating the secretion of the composition,or a mixture thereof into preparations which can be usedtherapeutically.

Nucleic acid molecules encoding a cupredoxin entry domain or a fusionprotein combining a either entry domain and a cargo compound can beinserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (Nabel et al., U.S. Pat. No. 5,328,4701994. USA), or by stereotactic injection (Chen et al., Proc Natl AcadSci USA, vol. 91, pp 3054-57 (1994)). The pharmaceutical preparation ofa gene therapy vector can include an acceptable diluent or can comprisea slow release matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

In one aspect, the composition is delivered as DNA such that the complexis generated in situ. In one embodiment, the DNA is “naked,” asdescribed, for example, in Ulmer et al., Science 259:1745-49 (1993) andreviewed by Cohen, Science 259 1691-92 (1993). The uptake of naked DNAmay be increased by coating the DNA onto a carrier, e.g. a biodegradablebead, which is efficiently transported into the cells. In such methods,the DNA may be present within any of a variety of delivery systems knownto those of ordinary skill in the art, including nucleic acid expressionsystems, bacterial and viral expression systems. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. See, e.g., WO90/11092, WO93/24640, WO93/17706, and U.S. Pat. No. 5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are non-essential for propagation inan appropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of the composition. In expression vectors, theintroduced DNA is operably-linked to elements such as promoters thatsignal to the host cell to transcribe the inserted DNA. Some promotersare exceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acomposition polynucleotide to an inducible promoter can control theexpression of the wt-azurin entry domain composition polypeptide orfragments. Examples of classic inducible promoters include those thatare responsive to α-interferon, heat shock, heavy metal ions, andsteroids such as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511(1990)) and tetracycline. Other desirable inducible promoters includethose that are not endogenous to the cells in which the construct isbeing introduced, but, however, are responsive in those cells when theinduction agent is exogenously supplied. In general, useful expressionvectors are often plasmids. However, other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, enhancer elements,promoters, and transcription termination sequences.

Kits Comprising a Cupredoxin Entry Domain-Cargo Compound Complex

In another aspect, the invention provides kits containing one or more ofthe following in a package or container: (1) a reagent comprising acomplex of a cupredoxin entry domain linked to a cargo compound; (2) areagent containing a pharmaceutically acceptable adjuvant or excipient;(3) a vehicle for administration, such as a syringe; (4) instructionsfor administration. Embodiments in which two or more of components(1)-(4) are found in the same container are also contemplated.

When a kit is supplied, the different components of the composition maybe packaged in separate containers and admixed immediately before use.Such packaging of the components separately may permit long-term storagewithout losing the active components' functions.

The reagents included in the kit can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized polypeptide orpolynucleotide, or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, etc., ceramic, metal or any other material typicallyemployed to hold similar reagents. Other examples of suitable containersinclude simple bottles that may be fabricated from similar substances asampules, and envelopes, that may comprise foil-lined interiors, such asaluminum or an alloy. Other containers include test tubes, vials,flasks, bottles, syringes, or the like. Containers may have a sterileaccess port, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to be mixed. Removable membranes may be glass,plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device, etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended embodiments.

EXAMPLES Example 1 Plasmid Constructions

Plasmids expressing fusion glutathione S-transferase (GST)-truncatedwt-azurin (azu) derivatives were constructed by a polymerase chainreaction using proofreading DNA polymerase. FIG. 6 shows a schematicrepresentation of various truncated wt-azurin constructs. For pGST-azu36-128, an amplified PCR fragment was introduced into the BamH1 andEcoR1 sites of the commercial GST expression vector pGEXSX (AmershamBiosciences, Piscataway, N.J. 08855). The fragment was amplified withpUC19-azu as a template and primers, 5′-CGGGATCC CCG GCA ACC TGC CGA AGAACG TCA TGG GC-3′(SEQ ID NO: 12) and 5′-CGGAATTC GCA TCA CTT CAG GGT CAGGG-3′ (SEQ ID NO: 13), where the additionally introduced BamHI and EcoRIsites are underlined respectively. Carboxyl-terminus truncation of azugene was cumulatively performed by introducing a stop codon usingQuickChange site-direct mutagenesis kit (Stratagene, La Jolla, Calif.92037).

For pGST-azu 36-50, pGST-azu 36-77 and pGST-azu 36-89, stop codons wereintroduced into Ser51, Ser78, and Gly90, respectively. The plasmidcarrying pGST-azu 36-128 was used as template DNA. Three sets ofoligonuclotides for site-direct mutagenesis are shown as follows. ForpGST-azu 36-50: 5′-GGC CAC AAC TGG GTA CTG TGA ACC GCC GCC GAC ATGCAG-3′ (SEQ ID NO: 14), and 5′-CTG CAT GTC GGC GGC GGT TCA CAG TAC CCAGTT GTG GCC-3′ (SEQ ID NO: 15). For pGST-azu 36-77: 5′-CCT GAA GCC CGACGA CTG ACG TGT CAT CGC CCA CAC C-3′ (SEQ ID NO: 16) and 5′-GGT GTG GGCGAT GAC ACG TCA GTC GTC GGG CTT CAG G-3′ (SEQ ID NO: 17). For pGST-azu36-89: 5′-CCA AGC TGA TCG GCT CGT GAG AGAAGG ACT CGG TGA CC-3′ (SEQ IDNO: 18), and 5′-GGT CAC CGA GTC CTT CTC TCA CGA GCC GAT CAG CTT GG-3(SEQ ID NO: 19). The plasmids pGST-azu 50-77 and pGST-azu 67-77 weregenerated by PCR using pGST-azu 36-77 as a template DNA.

Amplified PCR fragments, azu 50-77 and azu 67-77, were obtained usingforward primers 5′-CGGGATCC TGA GCA CCG CCG CCG ACA TGC AGG G-3′ (SEQ IDNO: 20) and 5′-CGGGATCC CCG GCC TGG ACA AGG ATT ACC TGA AGC CCG-3 (SEQID NO: 21), where the additionally introduced BamHI site is indicated byunderlining. The reverse primer, 5′-CGGAATTC GCA TCA CTT CAG GGT CAGGG-3′ (SEQ ID NO: 22), was utilized in both cases.

The plasmid carrying gst-azu 50-77 was used for generating pGST-azu50-66 by introduction of a stop codon in Gly67 using oligonuclotides asfollows: 5′-GAC GGC ATG GCT TCC TGA CTG GAC AAG GAT TAC C-3′ (SEQ ID NO:23), and 5′-GGT AAT CCT TGT CCA GTC AGG AAG CCA TGC CGTC-3′ (SEQ ID NO:24). The green fluorescent protein gene (gfp) encoding the greenfluorescent protein was also amplified by PCR. Forward and reverseprimers used were 5′-CGGGATCC CCA TGG TGA GCA AGGGCG-3′ (SEQ ID NO: 25)and 5′-CGGAATTC CTT GTA CAG CTC GTC CAT GCC G-3′ (SEQ ID NO: 26)containing BamHI and EcoRI sites at the 5′ end of each oligonuclotides.The resultant PCR fragment was ligated into the pGEXSX vector forcreating pGST-GFP. For the preparation of plasmid DNA carryinggst-gfp-azu 50-77, the azu 50-77 gene was amplified by PCR with pGST-azu50-77 as a template and primers 5′-CCGCTCGAG CCT GAG CAC CGC CGC CATGCAGGG-3′ (SEQ ID NO: 27) and 5′-TTTTCCTTTTGCGGCCGC TCA GTC GTC GGG CTT CAGGTA ATC C-3′ (SEQ ID NO: 28), where the introduced Xho I and Not I sitesare underlined respectively. Purified azu 50-77 fragment was introducedinto pGST-GFP at Xho 1 and Not 1 unique restriction enzyme sites

Example 2 Purification of Proteins

Wt-azurin and M44KM64E mutant azurin were prepared and purified asdescribed by Yamada, T. et al. Proc. Natl. Acad. Sci. USA, vol. 101, pp.4770-75 (2004), and in copending U.S. patent application Ser. No.10/720,603, the contents of which are incorporated by this reference.Briefly, the wt-azurin gene was amplified by PCR according to the methoddescribed by Kukimoto et al., FEBS Lett, vol. 394, pp 87-90 (1996). PCRwas performed using genomic DNA from P. aeruginosa strain PAO1 as atemplate DNA.

The amplified DNA fragment of 545 bp, digested with Hind111 and Pst1,was inserted into the corresponding sites of pUC19 so that the azuringene was placed downstream of the lac promoter to yield an expressionplasmid pUC19-azuA. E. coli JM109 was used as a host strain forexpression of the azurin gene. The recombinant E. coli strain wascultivated in 2YT medium containing 50 μg ml⁻¹ ampicillin, 0.1 mM IPTG;and 0.5 mM CuSO₄ for 16 h at 37° C. to produce azurin.

For preparation of the M44KM64E mutant azurin, site-directed mutagenesisof the azurin gene was performed using a QuickChange site-directedmutagenesis kit (Stratagene, La Jolla, Calif.). Mutations were confirmedby DNA sequencing.

Plasmid DNA, pET9a carrying the rus gene encoding the cupredoxinrusticyanin from Acidithiobacillus ferrooxidans, was obtained from Dr.Kazuhiko Sasaki, Central Research Institute of Electric Power Industry,Chiba, Japan.

Rusticyanin was isolated from E. coli BL21 (DE3) harboring the rus geneusing the method of Sasaki, K., et al. Biosci. Biotechnol. Biochem.,vol. 67, pp. 1039-47 (2003) with some modifications. Briefly, aceticacid buffer (pH 4.0) and CM-Sepharose (Sigma Chemicals, St. Louis, Mo.63178) were used instead of beta-alanin buffer (pH 4.0) and TSK-gelCM-650 column (Tosoh Bioscience, LLC, Montgomeryville, Pa. 18936). Twoother purified cupredoxins, plastocyanin from Phormidium laminosum andpseudoazurin from Achromobacter cycloclastes were obtained from Dr.Beatrix G. Schlarb-Ridley, University of Cambridge, UK and Dr.Christopher Dennison, University of Newcastle Upon Tyne, UK,respectively.

All recombinant GST-fusion derivatives were purified as follows: E. coliBL21 cells were used as the host strain. After induction with 0.4 mMIPTG at early log phase of growth in L broth, GST-fusion proteins werepurified from cell extracts by using Glutathione Sepharose 4B affinitychromatography and Sephadex 75 gel-filtration column with PBS (AmershamBiosciences, Piscataway, N.J. 08855). Purified proteins, wt azurin andGST-derivatives or other cupredoxins, labeled with ALEXA FLUOR®(Molecular Probes, Inc., Eugene, Oreg. 97402) were isolated according tomanufacturer's instructions. Unbound free fluorescent chemical wasremoved by gel-filtration column.

Example 3 Cell Cultures

J774 and UISO-Mel-2 cells (available from Frederick Cancer Research andDevelopment Center, Frederick, Md. U.S.A.) were cultured as described inYamada, T. et al. Infect. Immun. vol. 70, pp. 7054-62 (2002); Goto, M.,et al. Mol. Microbiol. vol. 47, pp. 549-59 (2003); and Yamada, T., etal. Proc. Natl. Acad. Sci. USA vol. 99, pp. 14098-103 (2002), thecontents of which are incorporated by reference. Human normal fibroblastcells (stock culture collection of the Department of Surgical Oncology,University of Illinois at Chicago (UIC), Chicago) were cultured in MEMwith Eagle's salt containing 2 mM L-glutamine, 0.1 mM MEM essentialamino acids and supplemented with 10% heat inactivated fetal bovineserum, 100 Units/ml penicillin and 100 μg/ml streptomycin. MCF-7 andMOF-10F cells were cultured as described in Punj et al. Oncogene23:2367-78 (2004).

Example 4 Co-Culture of J774, UISO-Mel-2 and Fibroblast Cells andConfocal Microscopy

J774, UISO-Mel-2, and fibroblast cells were cultured on individual coverslips. After overnight incubation, the cells were washed with freshmedia and all three cell lines were placed on a culture dish containing200 μg/ml of wt-azurin conjugated with ALEXA FLUOR® 568. The cells werethen incubated for 0.5 or 3.5 h at 37° C. under 5% CO₂.

For preparation of microscope samples, cells were cultured oncover-slips overnight at 37° C. Cultured cells were placed at 37° C. or4° C. for 2 h before protein treatment. Pre-warmed 37° C. fresh media orice-cold 4° C. fresh media were mixed with red-fluorescent (labeled withALEXA FLUOR® 568) cupredoxins or GST-fusion derivatives, and incubatedwith the cells. The cells were washed with PBS, and fixed with methanolat −20° C. for 5 min. After washing with PBS twice and the addition ofmounting media containing 1.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI)for staining nuclei (VECTASHILD, Vector, Burlingame, Calif.), imageswere taken by a confocal microscope.

Example 5 Entry of Cupredoxins into J774 Cells

Wt-azurin, its mutant variant M44KM64E, plastocyanin, pseudoazurin andrusticyanin were incubated with J774 cells as in Example 4 and the cellsexamined using confocal microscopy. In these experiments, thecupredoxins were conjugated with ALEXA FLUOR® 568 to fluoresce red andincubated with the J774 cells for 1 hr at 37° C. at a concentration of200 μg/ml, and in a separate experiment wild type azurin and rusticyaninwere incubated with J774 cells for 1 hr at 37° C. at a concentration ofabout 6 to 7 μM. The nucleus was stained blue with DAPI. A controlwithout the proteins was maintained. In all cases, the cupredoxins wereseen to enter into the cytosol of J774 cells. In similar experiments,auracyanin A and B enter preferentially to MCF7 cancer cells and notnon-cancerous control cells.

Example 6 Entry of Wt-Azurin and Rusticyanin into Various Cell Types

Wt-azurin exhibits a reduced cytotoxic activity towards MCF-10F cells ascontrasted with the MCF-7 cells. Punj et al. Oncogene 23:2367-2378(2004). J774, peritoneal macrophages, mast cells, human breast cancerMCF-7 and human normal epithelial MCF-10F cells (stock culturecollection of the Department of Surgical Oncology, University ofIllinois at Chicago (UIC), Chicago) were treated and examined as inExample 5 and tested to determine if wt-azurin could enter such cells.

Wt-azurin was internalized in J774 cells during 45 mm incubation.However, it was internalized very inefficiently in peritonealmacrophages or mast cells. Even after 6 hr incubation, such cells showedonly limited entry. Similarly, while wt-azurin entered the breast cancerMCF-7 cells efficiently, it showed an extremely reduced rate of entry inthe normal mammary MCF-10F cells.

Alexa Fluor®-conjugated azurin entered efficiently in UISOMel-2 andMCF-7 cancer cells but not in the normal mammary MCF 10A1 cells. AlexaFluor®-conjugated rusticyanin, however, not only entered the cytosol ofUISO-Mel-2 and MCF-7 cancer cells, but also in the normal MCF 10A1cells. Unlike in the cancer cells where rusticyanin was evenlydistributed in the cytosol, in MCF10A1 cells, much of the rusticyaninwas sequestered in the perinuclear space surrounding the nucleus.

Example 7 Wt Azurin-Mediated Cytotoxicity and Growth Inhibition

To further assess the specificity of entry of wt-azurin in variouscells, we determined the entry of Alexa fluor-conjugated wt-azurin inJ774, UISO-Mel-2 and normal fibroblast cells during incubation at 37° C.for 30 min and 3.5 hr. Wt-azurin was seen to enter rapidly in J774 andUISO-Mel-2 cells in 30 mm; very little wt-azurin was seen in the cytosolof fibroblasts during this period. After 3.5 hr of incubation, onlysmall amounts of wt-azurin were found in the fibroblasts.

A 3(4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide) (MTT) assay wasperformed for the measurement of the cytotoxicity of wt-azurin asdescribed by Yamada, T., et al. Infect. Immun. 70:7054-62 (2002), Goto,M., et al. Mol. Microbiol 47:549-59 (2003), and in co-pending U.S.patent application Ser. No. 10/720,603, filed Nov. 24, 2003, thecontents of which are incorporated by reference. FIG. 1( b) shows thatsignificant wt-azurin-mediated cytotoxicity was observed only with J774and UISO-Mel-2 cells during 24 hr incubation.

M44KM64E mutant azurin showed very little apoptosis-inducing activity inJ774 cells but at 1 mg/ml concentration significantly inhibited (about95%) cell cycle progression at the G₁ to S phase. Cell cycle progressionwas analyzed by flow cytometry, as described by Hiraoka, Y. et al.,Proc. Natl. Acad. Sci. USA, vol. 101:6427-32 (2004) and Yamada, T. etal. Proc. Natl. Acad. Sci. USA 101:4770-75 (2004), the contents of whichare incorporated by reference. FIG. 1( a) shows that when thefibroblasts were treated with 500 μg/ml or 1 mg/ml of M44KM64E mutantazurin, the extent of inhibition of cell cycle progression was about20%.

Example 8 Microinjection of Wt-Azurin into Fibroblast and MCF-10F Cells

Wt-azurin was microinjected into fibroblast and MCF-10F cells as usingthe method described by Punj, V., et al., Oncogene 23:2367-78 (2004).Cells were examined for induction of apoptosis, leading to nuclear DNAcondensation and fragmentation. Significant nuclear DNA (labeled bluewith DAPI) condensation and fragmentation were observed in microinjectedsingle cells after 5 hr incubation with wt-azurin, but not during a 30min. incubation with azurin.

Example 9 Internalization of Wt-Azurin Fusion Derivatives at 37° C.

A series of GST fusions of wt-azurin truncated at both the N- and theC-terminal were prepared and purified as in Example 1 (FIGS. 2( a) and2(b)). Using ALEXA FLUOR® 568 conjugated wt-azurin, GST and GST-azufusion derivatives, internalization in J774 cells at 37° C. during 1 hrincubation was examined using the method described in Example 5. Thenucleus was stained blue with DAPI.

While wt-azurin was internalized, GST remained at the periphery of thecells and was not internalized. GST-azu 36-128 and GST-azu 36-89 wereinternalized, as was GST-azu 36-77. Further truncations, however,demonstrated that while GST-azu 50-77 was internalized, GST-azu 36-50was highly inefficient and appeared to form clumps on the surface.

Example 10 Internalization of Azurin Fusion Derivatives at 4° C.

Internalization of wt-azurin and the GST-azu fusion derivatives in J774cells incubated at 4° C. was examined. At 4° C., internalization ofwt-azurin inside J774 cells during 1 hr incubation was severelyimpaired. Similar impairment was also seen with GST-azu 36-128 andGST-azu 36-89. The shorter GST-azu 36-77, GST-azu 50-77, GST-azu 50-66and GST-azu 67-77 demonstrated severe impairment of internalization at4° C.

Example 11 Energy-Dependent Internalization of the GST-GFP-Azu 50-77Fusion Protein in J774 and Melanoma UISO-MeI-2 Cells

GST was fused with GFP to make a GST-GFP fusion derivative.Additionally, azu 50-77 was fused to the GST-GFP (M_(r) 53 kDa) fusionprotein (FIG. 3( a)). The mobility of the purified GST, GST-GFP andGST-GFP-azu 50-77 fusion derivatives was examined on SDS-PAGE (FIG. 3(b)). Detection was by Coomassie Blue staining and Western blotting usinganti-azurin antibody (FIG. 3( c))

Flow cytometric determination of J774 cells treated with varyingconcentrations of GST-GFP showed that this protein does bind to J774cells. Flow cytometric separation of J774 cells treated with increasingconcentrations of GST-GFP-azu 50-77 fusion protein demonstratedsignificantly reduced fluorescence than GST-GFP alone (FIG. 4). It is tobe noted that internalization of GFP in mammalian cells is known to leadto loss of fluorescence. This reduction of fluorescence is also apparentwhen J774 cells are treated with 200 μg/ml of GST-GFP-azu 50-77 fusionprotein and incubated for increasing periods of time at 37° C.

To determine if there is any difference in the binding andinternalization profile of GST-GFP and GST-GFP-azu 50-77, both J774 andUISO-Mel-2 cells were incubated with GST-GFP and GST-GFP-azu 50-77 at37° C. and at 4° C. The green fluorescence was localized using confocalmicroscopy. In J774 cells, GST-GFP fusion protein bound to the surfaceand was not internalized both at 37° C. and at 4° C. In contrast,GST-GFP-azu 50-77 was found to be internalized at 37° C., but not at 4°C. In UISO-Mel-2 cells, the GST-GFP fusion protein was retained on thesurface both at 37° C. and at 4° C. In contrast, similar to J774 cells,GST-GFP-azu 50-77 fusion protein was seen to be internalized at 37° C.but not at 4° C.

Example 12 Wt-Azurin Entry into Mammalian Cells by a Cell MembranePenetration and an Endocytic Mechanism

If wt-azurin entry is solely dependent on receptor-mediated endocytosis,it could be blocked by protonophore carbonyl cyanide mchlorophenylhydrazone (CCCP), a mitochondrial uncoupler of energygeneration, or preincubation with unlabeled azurin or other cupredoxinsthat block the receptors. J774 and UISO-MeI-2 cells were incubated withthe cupredoxins at 10 fold excess concentration for 2 hr at 4° C., thecells washed thoroughly to remove the cupredoxins, and incubated withALEXA FLUOR® 568-conjugated azurin for 1 hr at 37° C. There was as muchinternalized azurin as in cells not treated with the cupredoxins. Theeffects of cytochalasin D (available from Sigma-Aldrich, St. Louis, Mo.63195), a known inhibitor of receptor-mediated endocytosis that disruptsthe cellular microfilament network, and Brefeldin A (available fromSigma-Aldrich, St. Louis, Mo. 63195), which is known to disrupt theGolgi apparatus and inhibit classical vesicle-mediated secretion, werealso tested. CCCP at 20 pM concentration significantly reduced theuptake of azurin in UISO-MeI-2 cells as did 0.25 to 0.5 pM cytochalasinD. Brefeldin A, on the other hand, had no significant effect.

Example 13 Entry of a GST-PEDIII-Azu 50-77 Fusion Derivative intoUISO-Mel-2 Cells

A GST-fusion of Pseudomonas aeruginosa exotoxin A domain III (PEDIII)was constructed as described by Hwang, J. et al., Cell 48:129-36 (1987);Reiter, Y. and Pastan, I., Trends Biotechnol. 16:513-20 (1998). ThisGST-PEDIII fusion derivative contained amino acids 381-613 of theexotoxin A. PEDIII is known to harbor ADP-ribosyl transferase activityand inhibits cellular protein synthesis in eukaryotic cells byinhibiting eukaryotic elongation factor 2.

Using PCR as described for the GST-GFP-azu 50-77, the azu 50-77 sequencewas introduced to the carboxyl end of the GST-PEDIII fusion protein(FIG. 5( a)). These two fusion proteins (GSTPEDIII and GST-PEDIII-azu50-77) were purified by glutathione-sepharose 4B column chromatographyas 52 and 54 kDa proteins (FIG. 5( b)). UISO-Mel-2 and normal fibroblast(FBT) cells were then incubated for 24 h at 37° C. with variousconcentrations of these proteins and the extent of cell death measuredby MTT assay as described in Example 7.

While GST-PEDIII demonstrated only low cytotoxicity, the GST-PEDIII-azu50-77 fusion protein had high cytotoxicity because of efficient entry toUISO-Mel-2 cells (FIG. 5( c)). In contrast, the fusion proteinsdemonstrated a low level of cytotoxicity towards the fibroblast cells.

Example 14 Destabilization of the α-Helix in wt-Azurin has NoSubstantial Effect on its Internalization in UISO-Mel-2 Cells

To examine if the α-helix plays a role in azurin entry, threehelix-destabilizing proline residues were introduced in positions 54, 61and 70 of wt-azurin (FIG. 6) and examined the entry of the full lengthA54PT61PK70P mutant azurin into UISO-Mel-2 cells. Single and doublemutations in these positions were also constructed and tested for entry.The A54PT61PK70P mutant azurin was prepared by site-directed mutagenesisof the azurin gene using the QuickChange site-directed mutagenesis kit(Stratagene, La Jolla, Calif.).

The mutants were incubated at 200 μg/ml with UISO-Mel-2 cells for 1 hrat 37° C., after which the fluorescence was localized by confocalmicroscopy. In all cases, the ALEXA FLUOR® 568-conjugated mutant azurinsentered UISO-Mel-2 cells. Similarly, when the GST-GFP-azu 50-77 fusionprotein, as well as its triple A54PT61PK70P azu mutant variant, wereexamined for entry in UISO-Mel-2 cells, no significant difference wasobserved.

Example 15 Entry of a GST-PEDIII-Rusticyanin Fusion Derivative intoUISO-Mel-2 Cells

A GST-fusion of Pseudomonas aeruginosa exotoxin A domain III (PEDIII)and was constructed as in Example 13. Using PCR as described for theGST-GFP-azu 50-77, full-length rusticyanin sequence was introduced tothe carboxyl end of the GST-PEDIII fusion protein. The fusion proteinwas purified by glutathione-sepharose 4B column chromatography.UISO-Mel-2 and FBT cells were then incubated for 24 h at 37° C. withvarious concentrations of the fusion protein and the extent of celldeath measured by MTT assays as described in Example 7.

The GST-PEDIII-rusticyanin fusion protein exhibited high cytotoxicityagainst UISO-Mel-2 cells (FIG. 7). In contrast, the fusion proteindemonstrated only a low level of cytotoxicity towards the FBT cells.

Example 16 Competition of Azurin with GST-Azu 55-77 for Entry into J774Cells

A competition experiment was performed with unlabeled azurin at 37° C.in presence of 7 μM Alexa Fluor®-conjugated GST-azu 50-77. J774 cellswere incubated without Alexa Fluor®-conjugated azurin, with AlexaFluor®-conjugated GST-azu 50-77 (7 μM) and with Alexa fluor-conjugatedGST-azu 50-77 (7 μM) in presence of 7, 14 and 56 μM of unlabeled azurinfor 1 h at 37° C. before determining GST-azu 50-77 entry. The resultsclearly demonstrated that in comparison to labeled 7 μM GST-azu 50-77alone, the presence of increasing amounts of unlabeled azurin (7, 14 and56 μM) increasingly competed with the entry of labeled 7 μM GST-azu50-77. In contrast, when similar concentrations of (6, 12 and 48 μM)unlabeled rusticyanin were used in presence of labeled GST-azu 50-77,very little effect on the entry of GST-azu 50-77 was seen.

Example 17 Sequence and Structural Comparison of Azurin ProteinTransduction Domain (PTD) with Other Cupredoxins

The sequence identity between azurin and rusticyanin in the azu 50-77region is less than 20%, as is true of several other cupredoxins (DeRienzo et al., Protein Science 9:1439-1454, 2000; Murphy et al., J. Mol.Biol. 315:859-871, 2002). A structural analysis using VAST algorithm(Gibrat et al., Curr. Opin. Struct. Biol. 6:377-385, 1996) betweenazurin and several members of the cupredoxin family demonstrated asignificant identity between azu 50-77 region and that of the cupredoxinauracyanin B, a cupredoxin from the green thermophilic photosyntheticbacterium C. aurantiacus (Bond et al., J. Mol. Biol. 306:47-67, 2001).Other members of the cupredoxin family, while demonstrating structuralsimilarity with other regions of azurin lacked significant identity withthe azurin PTD, amino acids 50-77 (FIG. 8( a)). Indeed, when comparedwith other proteins whose structures have been deposited in the proteindatabase, there was very little structural similarity between the azuPTD and other proteins.

A multiple amino acid sequence alignment of the residues in the P.aeruginosa PTD region with known sequences of other bacterial azurinsfrom pathogens using the CLUSTAL X alignment program (Higgins and Sharp,id. 1988). While the phytopathogenic P. syringae azurin showed highidentity with P. aeruginosa azurin PTD region, an azurin-like proteinfrom Neisseria meningitidis (Gotschlich and Seiff, FEMS Microbiol. Lett.43:253-255 (1987); Kawula et al., Mol. Microbiol. 1:179-185 (1987);Cannon, Clin. Microbiol. Rev. 2:S1-S4 (1989)) also showed significantidentity with the PTD domain of P. aeruginosa azurin, as did azurinsfrom Vibrio parahaemolyticus and Bordetella bronchiseptica (FIG. 8( b)).A motif sequence D-G-X—X—X—X—X-D-X—X—Y—X—K—X—X-D (SEQ ID NO: 35) wasfound conserved in all these azurins.

Example 18 N. meningitidis Laz (H8-Azurin) Induces Cell Death in theBrain Tumor Cell Line LH229

Neisseria meningitidis (Nm) causes cerebrospinal meningitis bydisseminating in the blood stream, crossing the blood brain barrier,presumably by a transcellular route through brain endothelial cells, andinvading the meninges. The azurins from both Pseudomonas aeruginosa andNeisseria meningitidis have similar structures and high amino acidsequence homology (>50%). In addition, the N. meningitidis azurin ispart of a longer polypeptide, Laz (SEQ ID NO: 30), which harbors in itsN-terminal a surface peptide called H.8, which is present in the outermembrane of N. meningitidis (Cannon, J. G., Clin. Microbiol. Rev. vol.2, pp. 51-54 (1989)). The surface-exposed H8 antigenic epitope at alsocarries a signal for lipidation. The complete N. meningitidis H8 outermembrane protein remains on the external surface of N. meningitidiscells.

The Neisseria gonorrhoeae Laz protein is very similar to the N.meningitidis Laz protein. The N. gonorrhoeae laz gene was cloned into E.coli, and then the laz gene in the E. coli was hyper-expressed toproduce the Laz protein (FIG. 13). The ability of the purified P.aeruginosa azurin and the purified N. gonorrhoeae Laz protein to inducecell death in the brain tumor cell line LH229 as measured by the MTTassay (Yamada, T., et al., Cell Cycle vol. 3, pp. 1182-1187 (2004)).

While P. aeruginosa azurin, when expressed in E. coli, is present in theperiplasm, the Laz protein, when expressed in E. coli, was found in theouter membrane of E. coli. Additionally, while azurin had very lowcytotoxicity towards the brain tumor cells within 24 hours, Laz showedhigh cytotoxicity, allowing the death of 90% of the brain tumor cells in24 hours (Table 3). Azurin did show increasing cytotoxicity over 48hours (Table 4). This experiment indicates that P. aeruginosa azurin andthe N. gonorrhoeae H8-azurin (Laz, SEQ ID NO: 36) will be useful todiagnose and/or treat brain tumors in vitro and in vivo.

If Laz reduces brain tumor growth in vivo, all or part of the proteinwill be used as the “cupredoxin entry domain” of the invention totransport cargo molecules, including fluorescent or radioactive tags ortumor-killing drugs/toxins, into the brain, and into brain tumor cellsfor diagnostic or therapeutic purposes.

TABLE 3 Cytotoxicity of P. aeruginosa azurin and N. gonorrhoeae Laz toBrain Cancer Cell Line LH229 After 24 Hours Incubation. Protein Conc.Cytotoxicity (%) STDEV P. aeruginosa azurin 0 0.00 0.00 N. gonorrhoeaeLaz 0 0.00 0.00 P. aeruginosa azurin 25 5.86 2.45 N. gonorrhoeae Laz 2518.80 1.26 P. aeruginosa azurin 50 5.48 2.43 N. gonorrhoeae Laz 50 23.591.57 P. aeruginosa azurin 100 6.00 4.13 N. gonorrhoeae Laz 100 41.021.49 P. aeruginosa azurin 200 8.27 4.79 N. gonorrhoeae Laz 200 78.992.06 P. aeruginosa azurin 400 7.25 3.69 N. gonorrhoeae Laz 400 94.690.87 P. aeruginosa azurin 800 13.22 7.67 N. gonorrhoeae Laz 800 98.360.62

TABLE 4 Cytotoxicity of P. aeruginosa azurin and N. gonorrhoeae Laz toBrain Cancer Cell Line LH229 After 48 Hours of Incubation. Protein Conc.(μg/ml) Cytotoxicity (%) STDEV P. aeruginosa azurin 0 0.00 0.00 N.gonorrhoeae Laz 0 0.00 0.00 P. aeruginosa azurin 25 11.65 1.07 N.gonorrhoeae Laz 25 20.04 4.25 P. aeruginosa azurin 50 8.55 2.51 N.gonorrhoeae Laz 50 25.52 1.80 P. aeruginosa azurin 100 16.19 2.60 N.gonorrhoeae Laz 100 39.72 0.92 P. aeruginosa azurin 200 16.30 1.91 N.gonorrhoeae Laz 200 87.01 1.21 P. aeruginosa azurin 400 61.96 19.15 N.gonorrhoeae Laz 400 98.75 0.96 P. aeruginosa azurin 800 82.71 5.91 N.gonorrhoeae Laz 800 99.93 0.12

Example 19 Cytotoxicity of the N. gonorrhoeae Laz Protein to Brain TumorCells

Several plasmids were constructed encoding protein constructs includingP. aeruginosa azurin, N. gonorrhoeae Laz (SEQ ID NO:36), and fusionproteins, and the proteins tested in the MTT assay with brain tumor celllines. The protein constructs tested include the N. gonorrhoeae Laz H.8region alone, P. aeruginosa azurin alone, N. gonorrhoeae azurin alone,and the constructs depicted in FIG. 10. The plasmids encoding theprotein constructs are transformed into E. coli or another suitableexpression system, and the protein constructs expressed and theresulting proteins purified. Brain tumor cell lines include NL229 andCCF-STTG1.

The results of this experiment indicate that the H.8 region and/or theazurin of the Laz gene are required to allow the protein construct to becytotoxic to brain tumor cells. This experiment also determines if otherazurins will be more cytotoxic to brain tumor cells if fused to the H.8region. The results of this experiment indicate that the H.8 region canadapt a cupredoxin to be cytotoxic to brain cancer cell by transportingthe attached azurin into the brain cancer cells. Accordingly, H.8 can beused as a transport domain to transport a cargo molecule(s) into braincancer cells. Useful cargos include cancer treatments and diagnosticagents as known in the art and set forth herein.

Example 20 Treatment of Patients Suffering from Cancer

A Phase I/II clinical trial of a cupredoxin entry domain-exotoxin Adomain III fusion (Study Drug) will be performed in patients sufferingfrom cancer. Specifically, the cupredoxin entry domain is the 50-67amino acid region from Pseudomonas aeruginosa and the cargo is theexotoxin A domain III from Pseudomonas aeruginosa, making the fusionprotein “PEDIII-azu 50-67.” This fusion protein will be constructed asillustrated in Example 13.

Forty-nine adult patients with histologically verified cancers of thebreast, colon and melanoma who demonstrate clinical and radiographicprogression or recurrence following adequate treatment by currentlyavailable FDA-approved chemotherapeutic drugs and regimen will beenrolled in an open-label prospective study administering the StudyDrug. To be eligible for enrollment in the study, all patientsdemonstrate increasing volume of measurable tumor after completion ofapproved course of chemotherapy regimens. The evidence of persistentmetastatic deposits and/or continued increase in size or volume must behistologically established. This histological proof can be obtained by afine needle aspiration (FNA) biopsy.

The treatment program will be instituted after obtaining informedconsent from all patients in accordance with the Institutional ReviewBoard of the University of Illinois, Chicago and the FDA. The patientswill have no intercurrent illness such as other malignancy, history ofprevious malignancy, blood dyscrasias, insulin dependent diabetes orother serious cardiovascular diseases which might interfere inappropriate evaluation of the effects of the proposed therapy. Baselineblood work (Complete Blood Counts [CBC] and Serum Chemistry) includingliver function studies (LFT) will be performed prior to initiation oftherapy. All eligible patients must not receive any cancer chemotherapyconcurrently during the period of the trial.

The study drug(s) will be administered by daily intravenous injection ofa pharmaceutically acceptable preparation of the Study Drug for 12 weeksand the subjects will be observed for any dose limiting toxicity. Therewill be 7 dose levels starting with 10 mg/kg/day and increasing by 5mg/kg/day up to a maximum dose of 50 mg/kg/day. The efficacy of eachdose level will be recorded in 7 patients with advanced measurablecancer (breast, colon, and melanoma).

The response will be estimated by measuring the measurable tumor in 2dimensions (a and b). 1) Total disappearance of the target metastatictumors will be considered as complete response (CR); 2) A 75% reductionwill be considered excellent, partial response (PR); and 3) A goodresponse (PR) will be post treatment reduction in size by 50%. 4)Reduction of 25% in size will be considered as stable disease (SD) and5) <25% will be considered as no response (NR). Patients demonstrating aprogression of disease will have their treatment discontinued but willbe followed for an additional 12 weeks.

Total disappearance, and any reduction in size of the target metastatictumors will indicate that the azurin treatment is effective for treatingcancer. Other indications that the azurin treatment is effective are adecrease rate of in the appearance of new metastatic tumors and adecrease in the angiogenesis associated with tumors.

Various modifications and variations of the described examples andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specificembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in related fields areintended to be within the scope of the following claims.

The invention claimed is:
 1. An isolated peptide, consisting of asequence that has at least about 90% amino acid sequence identity toless than a full-length wild-type cupredoxin or H.8 outer membraneprotein, and which facilitates the entry of a linked molecule into amammalian cancer cell, and wherein the cupredoxin or H.8 outer membraneprotein is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36 andSEQ ID NO:
 43. 2. The peptide of claim 1, which is at least about 10residues and not more than about 50 residues in length.
 3. The peptideof claim 1, comprising a sequence which has at least about 90% aminoacid sequence identity to a sequence selected from the group consistingof SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ IDNO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:46, and SEQ ID NO: 47.4. The peptide of claim 3, comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, and SEQID NO:
 47. 5. The peptide of claim 3, consisting of a sequence selectedfrom the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ IDNO: 46, and SEQ ID NO:
 47. 6. The peptide of claim 1, comprising anamino acid sequence selected from the group consisting ofDGXXXXXDXXYXKXXD (SEQ ID NO: 35) and DGXXXXDXXYXKXXD (SEQ ID NO: 48);wherein D is aspartic acid, G is glycine, Y is tyrosine, K is lysine andX is any amino acid.
 7. A complex comprising a cargo compound and apeptide, wherein the peptide has at least about 90% sequence identitywith a cupredoxin, or a fragment thereof, wherein the peptide, orfragment thereof is linked to the cargo compound, and wherein thepeptide facilitates entry of the cargo compound into a mammalian cancercell, and wherein the cupredoxin is selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 36 and SEQ ID NO:
 43. 8. The complex of claim 7, whereinthe peptide consists of a sequence that has at least about 90% aminoacid sequence identity to less than a full-length wild-type cupredoxinor H.8 outer membrane protein, and which facilitates the entry of alinked molecule into a mammalian cancer cell, wherein the cupredoxin orH.8 outer membrane protein is selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 36 and SEQ ID NO:
 43. 9. The complex of claim 7, wherein thecargo compound is selected from the group consisting of a polymer ofamino acid residues, lipoprotein, polysaccharide, nucleic acid, dye,microparticle, nanoparticle, toxin and drug.
 10. The complex of claim 9,wherein the cargo compound is a polymer of amino acid residues that islinked to the cargo compound to form a fusion protein.
 11. The complexof claim 7, wherein the cargo compound is a toxin.
 12. The complex ofclaim 11, wherein the toxin is Pseudomonas aeruginosa exotoxin A or afragment thereof.
 13. The complex of claim 7, wherein the cargo compoundis a detectable substance.
 14. The complex of claim 13, wherein thedetectable substance is detectable by a method selected from the groupconsisting of fluorimetry, microscopy, X-ray CT, MRI and ultrasound. 15.A pharmaceutical composition comprising the complex of claim 7 with apharmaceutically suitable carrier.
 16. A method comprising contacting acell or cells with the complex of claim
 7. 17. The method of claim 16,wherein the cell or cells originate from a patient suffering fromcancer, and further comprising reintroducing the cell or cells into thepatient.
 18. The method of claim 16, wherein the cell is a cancer cell.19. The method of claim 18, wherein the cell is a cancer cell selectedfrom the group consisting of osteosarcoma cell, lung carcinoma cell,colon carcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcomacell, breast carcinoma cell, liver carcinoma cell, bladder carcinomacell, melanoma cell, brain tumor cell and prostate carcinoma cell.
 20. Amethod of treating a patient with cancer, wherein the complex of claim 7is administered to said patient in a therapeutically effective amount.21. The method of claim 20, wherein the complex is administered in amanner selected from the group consisting of intravenously, topically,subcutaneously, intramuscularly, and into tumor.
 22. The method of claim20, wherein the complex is co-administered with another cancertreatment.
 23. A method for imaging cancer in a patient, wherein thecomplex of claim 13 is administered to said patient, and the location ofthe cargo compound is detected.
 24. The method of claim 23, wherein thecargo compound is an X-ray contrast agent and the location of the cargocompound is detected by X-ray CT.
 25. The method of claim 23, whereinthe cargo compound is a magnetic resonance imaging contrast agent andthe location of the cargo compound is detected by MRI.
 26. The method ofclaim 23, wherein the cargo compound is an ultrasound contrast agent andthe location of the cargo compound is detected by ultrasound imaging.27. A method for diagnosing cancer, wherein a cell is contacted with thecomplex of claim 13 and the location of the cargo molecule is detected.28. A kit comprising a reagent comprising the complex of claim
 7. 29.The kit of claim 28, further comprising a reagent comprising apharmaceutically-acceptable adjuvant or excipient.
 30. The kit of claim28, further comprising a vehicle for administration of the reagent. 31.The peptide of claim 1, wherein the structure of the peptide is modifiedto extend or optimize the half life of the peptide in the bloodstream.32. A nucleic acid molecule, which encodes the peptide of claim
 1. 33. Anucleic acid molecule, which encodes the complex of claim
 10. 34. Themethod of claim 23, wherein said peptide comprises a sequence selectedfrom the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO:
 47. 35. The method ofclaim 27, wherein said peptide comprises a sequence selected from thegroup consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 45, SEQ ID NO: 46, and SEQ ID NO:
 47. 36. The peptide of claim 1,wherein said full-length wild-type cupredoxin or H.8 outer membraneprotein is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, andSEQ ID NO: 43.